Our long-term goal is to develop non-invasive, optical technologies to monitor the functional development of engineered tissues in vitro and in vivo. The objective of this application is to develop optical biomarkers based on endogenous sources of optical contrast that obviate the use of exogenous stains and can be used to report quantitatively on the biochemical and structural composition of engineered tissues. The proposed studies focus on the characterization of adipose and bone engineered tissues developed from silk scaffolds seeded with human mesenchymal stem cells. The central hypothesis of the application is that linear and non-linear depthresolved imaging methods based on the natural light scattering and fluorescence signatures of cell and matrix components of engineered tissues can be developed to report on the dynamic changes that occur prior to and following implantation of engineered tissues. Our hypothesis is based on preliminary evidence acquired from in vitro samples, which indicate that endogenous optical signals can be used to monitor changes in the biochemistry and morphology of differentiating stem cells, silk scaffolds and deposited collagen. The rationale for the proposed research is that the establishment of non-invasive methods that allow monitoring of the dynamic changes that occur within engineered tissues will play an essential role in the development and optimization of innovative, functional engineered tissue constructs. To achieve our goal we will characterize the endogenous fluorescence and light scattering signals from different cell and matrix components of engineered tissues developed in vitro (Aim 1). We will develop a system that will optimize acquisition of these optical signals from animals (Aim 2) and we will use these biomarkers to characterize non-invasively the integration of these engineered tissues in vivo following implantation either within a mammary fat pad or a cavarial bone defect mouse model (Aim 3). This will be the first time that dynamic monitoring of the biochemical and structural function of implanted engineered tissues is achieved in vivo using non-invasive means based on endogenous optical signals. This proposal is highly relevant to the improvement of public health as it will enable the efficient development of functional bone and adipose engineered tissues. Thus, millions of patients that undergo surgical procedures for the repair or reconstruction of such tissues will ultimately benefit from this work. Public Health Relevance Statement (provided by applicant): This proposal is highly relevant to the improvement of public health as it will enable the efficient development of functional bone and adipose engineered tissues. Thus, millions of patients that undergo surgical procedures for the repair or reconstruction of such tissues will ultimately benefit from this work.